Production and bioprocessing of epothilone B from Aspergillus niger, an endophyte of Latania loddegesii, with a conceivable biosynthetic stability: anticancer, anti-wound healing activities and cell cycle analysis

Epothilones are one of the common prescribed anticancer drugs for solid tumors, for their exceptional binding affinity with β-tubulin microtubule, stabilizing their disassembly, causing an ultimate arrest to the cellular growth. Epothilones were initially isolated from Sornagium cellulosum, however, their extremely slow growth rate and low yield of epothilone is the challenge. So, screening for a novel fungal endophyte dwelling medicinal plants, with higher epothilone productivity and feasibility of growth manipulation was the objective. Aspergillus niger EFBL-SR OR342867, an endophyte of Latania loddegesii, has been recognized as the heady epothilone producer (140.2 μg/L). The chemical structural identity of the TLC-purified putative sample of A. niger was resolved from the HPLC, FTIR and LC–ESI–MS/MS analyses, with an identical molecular structure of the authentic epothilone B. The purified A. niger epothilone B showed a resilient activity against MCF-7 (0.022 μM), HepG-2 (0.037 μM), and HCT-116 (0.12 μM), with selectivity indices 21.8, 12.9 and 4, respectively. The purified epothilone B exhibited a potential anti-wound healing activity to HepG-2 and MCF-7 cells by ~ 54.07 and 60.0%, respectively, after 24 h, compared to the untreated cells. The purified epothilone has a significant antiproliferative effect by arresting the cellular growth of MCF-7 at G2/M phase by ~ 2.1 folds, inducing the total apoptosis by ~ 12.2 folds, normalized to the control cells. The epothilone B productivity by A. niger was optimized by the response surface methodology, with ~ 1.4 fold increments (266.9 μg/L), over the control. The epothilone productivity by A. niger was reduced by ~ 2.4 folds by 6 months storage as a slope culture at 4 °C, however, the epothilone productivity was slightly restored with ethylacetate extracts of L. loddegesii, confirming the plant-derived chemical signals that partially triggers the biosynthetic genes of A. niger epothilones. So, this is the first report emphasizing the metabolic potency of A. niger, an endophyte of L. loddegesii, to produce epothilone B, that could be a new platform for industrial production of this drug. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-024-02495-x.


Introduction
Epothilones, a group of 16-membered natural macrolides with the molecular structures including epoxide, thiazole and ketone moieties, that was firstly recovered from Sorangium cellulosum [40].Naturally, Epothilone is a hybrid metabolite of polyketides and non-ribosomal peptide that was has been synthesized by the polyketides synthase type I and non-ribosomal peptide synthetase [13].Epothilones have a strong activity against several solid human cancer cells, especially the drugsresistant cells [42].The strong antiproliferative efficiency of epothilones elaborated from their high binding affinity with β-tubulin, provoking tubulin polymerization and microtubule stability [8,11].Microtubules are polymers of α, β-tubulin dimers, with unique dynamic biological function that has been recognized as a susceptible target to the antimitotic drugs [17].By targeting microtubules, the antimitotic drugs disrupt the dynamics of cellular multiplication, stopping the G2/M phase of the cell cycle, provoking the apoptosis and ultimate cell death [35].Two classes of the microtubule targeting agents were recognized; microtubule-stabilizing "microtubule polymerization enhancers" (Epothilones, Taxol), and microtubule-destabilizing compounds "microtubule polymerization inhibitors" (Vinca alkaloids, colchicin) [37,75,79].Three sites on α, β-tubulin subunits were identified for binding with Taxol [6,32], vincristine [73] and colchicine [48].Epothilones and Taxol have the same recognition site on the microtubules, suppressing the growth of tumor cell by the same mechanism via induction of the microtubules polymerization [13,69].However, epothilones have a powerful activity to halt the growth of Taxol-resistant tumors, by inhibiting the drug efflux pump P-glycoprotein as multidrug-resistance associated protein, or tubulin mutations of tumor cells [7,64].In addition, epothilones have an affordable biological activity than Taxol owing to their remarkable water solubility, so no need for adjuvants for the applications, and lower binding energy with tubulin, compared to Taxol.So, epothilones exhibited an antiproliferative activity by more than 1000 folds higher than Taxol against different drug-resistant tumors "expressing P-glycoprotein" [3].Currently, epothilones have been produced by S. cellulosum, nevertheless, the unusually slow rate of growth, and low yield of epothilones are the prominent restricting reason halting this approach to be a commercial platform for Epothilone production [75].Recently, Aspergillus fumigatus, was confirmed to have the biosynthetic potency to produce Epothilone B, by the same antiproliferative activity, chemical structural identity of authentic one of S. cellulosum [29].The overall yield of epothilone by the nutritionally optimized A. fumigatus was actually higher than that of Burkholderia by approximately 6.2 folds (55 μg/g biomass) [51].However, the decrease of epothilone productivity with the preservation and sub-culturing of A. fumigatus is the main challenge, so, screening for novel isolates of a plausible metabolic stability of epothilone production is our main objective.The wide array of medicinal plants possessing ethnopharmacological and pharmaceutical attributes may harbor a multitude of novel endophytic fungal isolates with unique bioactive metabolites [27].The family Arecaceae has been frequently recognized with their ethnopharmacological properties, having diverse bioactive metabolites [68].Among the members of this family, Cocos nucifera, Sabal bermudana, Livistona chinensis and Wallichia caryotoides were frequently studied regarding to their endophytic fungal flora and their metabolic activities [36,70].Latania loddegesii, is one of the most common species of family Arecaceae, however, there is no report about the ethnopharmacological activity and biological identity of their endophytic microorganisms.So, the main objective of this work was to recover a novel fungal endophyte of Latania loddegesii, with higher epothilone productivity and reliable biosynthetic stability with storage.

Collection of plant samples and isolation of the endophytic fungi
Latania loddegesii belongs to the family Arecaceae from El-Abd Garden in Giza, Egypt in February 2022, was obtained and taxonomically identified by Therese Labib, a Plant Taxonomist.The herbarium specimen voucher of identification code ZU-Ph-Cog-0515 has been conserved at the Pharmacognosy Department Herbarium, Zagazig University.The plant sample was transported in plastic aseptic bags to the lab, rinsed by sterile water, and the fungal endophytes were isolated [27].Briefly, the plant leaves were cut to small sections of 1 × 1 cm, sterilized with 70% ethanol, and 2.5% sodium hypochlorite for 2 min [20,31,[26][27][28].Subsequently, the plant sections were placed on the surface of potato dextrose agar (PDA) (200.0 g potato extract, 20.0 g glucose, and 20.0 g agar per liter), with ampicillin at 1 μg/ml, then the cultures were incubated at 30 °C, for 15 days [29].The distinct colonies of fungi were collected, purified, stored as slope cultures.The isolated fungi were identified based on their morphological features according to the universal keys [9,16,59,62,63].The microscopical features of the potent fungal isolates were visualized by Scanning Electron Microscope (JEOL JSM 6510 l), at 20.00 kv [43].

Screening for epothilone production by the recovered fungal isolates
The fungal isolates of L. loddegesii were screened for their epothilone producing potency [29].Briefly, two plugs of 6-day old of each fungal isolate were inoculated to 50 ml of potato dextrose broth medium in 250 mL/Erlenmeyer conical flask, then incubated at 30 °C for 10 days.Triplicates of each isolate were prepared.The culture media were filtered, the filtrate was centrifuged at 5000 rpm, the clear supernatant was extracted with equal volume of ethyl acetate, and the extract was concentrated by rotary evaporator at 50 °C, till minimum volume (~ 2 ml), then stored at 4 °C.The ethyl acetate extracts were checked by TLC (Merck 1 mm (20 × 20 cm), Silica gel 60 F254, Darm., Germ.) using methylene chloride and methanol (95:5) as a developing system [8,40].The TLC plates were visualized by UV illumination (MinUVIS, Heid.Germany) at λ 254 nm, and the developed blue colored spots of the same mobility rate, were considered normalized to the authentic Epothilone B (Cat#0.152044-54-7).The recognized spots of silica gel on the TLC plates were scraped off, dissolved in methylene chloride and the epothilone samples were eluted [8,29,40].The purity and concentration of the extracted epothilone was measured by HPLC (Agilent Technology, G1315D) of RP-C18 column (Cat.#959963-902) using methanol, acetonitrile and water (25:35:40, v/v/v) as an isocratic mobile phase, at flow rate 1 ml/min for 25 min and photodiode array detector [40,50,54,77].The purity and concentration of assumed epothilone B were verified from the retention time, and area under the peak compared to authentic one (Cat#0.152044-54-7)at λ 254 nm [40].

Molecular identification of the selected fungi
The most epothilone producing fungi was verified from the sequence of their ITS region [1,31,26,29,56].Genomic DNA (gDNA) was extracted by cetyltrimethylammonium bromide reagent, and their concentration were assessed by 1.5% agarose gel [24].The DNA was used as a PCR template, with the primers; ITS4 5′-GAA GTA AAA GTC GTA ACA AGG-3′ and ITS5 5′-TCC TCC GCT TAT TGA TAT GC-3′, The PCR reaction had 2 μl of gDNA, primers (10 pmol), and 10 μl of 2 × PCR master mixture (Cat.#25,027), and completed to 20 μl with sterile water.The PCR was programed to initial denaturation 95 °C for 2 min, followed by denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 5 min.The PCR products were analyzed by 2% agarose gel in 1 × TBE buffer, compared to DNA ladder (Cat.#PG010-55DI).The amplicons were purified, and sequenced by the Applied Biosystems Sequencer, and the retrieved sequence was non-redundantly annotated by BLAST on the NCBI.The phylogenetic relatedness of the sequence was analyzed by ClustalW muscle algorithm at 100 bootstrap replications, with the neighbor-joining tool by MEGA X [71].

Chemical structure validation of the purified epothilone
The functional groups of putative epothilone from the fungal isolate were resolved by FT-IR Spectrometer within the 400-4000 cm −1 , in KBr pellets, and the UV/ Vis spectra were measured at λ 200-600 nm (RIGOL Spectrophotometer).The identity of putative Epothilone was determined by LC-ESI-MS/MS, using Hypersil Gold C18 column, LCQ Deca mass spectrometer with electrospray source, in a positive mode.The mobile phase A composed of water of 0.1% formic acid, whereas phase B consisted of acetonitrile in 0.1% formic acid.Over a period of 30 min, a gradient of solution B ranging from 2 to 98% was applied, at a flow rate of 0.2 ml/min.The selected molecular mass was further fragmented by MS/ MS, and the molecular pattern of fragmentation was annotated compared to reported data [29].

Anticancer activity of the ethyl acetate extract and purified epothilone
The antiproliferative activity of ethyl acetate extract, and putative epothilone from the selected fungus was assessed against colon carcinoma cells (HCT-116), breast carcinoma (MCF-7), and hepatocellular carcinoma (HepG-2), in addition to normal Vero cells by MTT assay [15].The tested cell lines were purchased from the ATCC, grown on RPMI medium (Dulbecco's Modified Eagle Medium, HEPES, GlutaMAX ™ ) of 10% FBS, in 96-microtiter plate.The plate was inoculated with 10 3 cells/ well, incubated under humidified atmosphere of 5% CO 2 for 12 h at 37 °C, then the tested compounds were amended to the wells at different concentrations, and the plates were re-incubated for a 48 h at the standard conditions.After incubation, 25 µL of the MTT reagent was amended, and the developed purpled-colored formazan complex was assessed at λ 570 nm.The IC 50 values were expressed by concentration of the putative epothilone reducing the initial growth of the tumor cells by about 50%, compared to control [15].To assess the effectiveness and selectivity of the extracted epothilone, the selectivity index (SI) value was assessed; SI = (CC 50 of a normal cell line (Vero)/ IC 50 of cancer cell line).The value of selectivity index (SI) exceed 3, signifies to the drug significant selectivity towards cancer cell lines [76].

Antifungal activity of the ethyl acetate extracts
The activity of the crude extracts of the recovered endophytic fungi was assessed against Aspergillus flavus, as a model human fungal pathogen [21].The tested fungi were cultured on potato dextrose broth (PDB), for 10 days at 28 °C, filtered, and the filtrates were extracted by ethyl acetate [29], the extracts (20 µl of 50 μg/ml) were loaded into 6 mm discs of Whatman #1 filter paper, placed on the surface of PDA culture of A. flavus [34].The plates were incubated at 28 °C for 5 days, using fluconazole and ethyl acetate as positive and negative controls.Triplicates of each experiment were prepared.To determine the MIC values, various concentrations of the tested extracts were loaded to the filter paper discs (1, 5, 10, 25, 50 and 100 μg/ml), positioned on surface of A. flavus PDA plate culture, then the diameters of the inhibition zones were determined.

Anti-wound healing efficiency of purified epothilone
The anti-wound healing activity of the purified A. niger epothilone towards the HepG-2, and MCF-7 cells was assessed [39,46].Briefly, the HepG-2 and MCF-7 cells were seeded at 5 × 10 6 cells to 12-well culture plate, incubated for 24 h at 37 °C, allowing to grow as confluent cellular monolayer, then a scratch in a straight line were done.After gently washing of cells, the wells were amended with a fresh medium of epothilone at IC 25 value, normalized to control culture (without epothilone), the plates were re-incubated as abovementioned conditions.The wound healing due to the cell migration was imaged, and the percentage of wound closure was assessed relied on the gap area of cells due to the epothilone, compared to control of DMSO (Conc.2%) treated cells.

Apoptosis analysis of MCF-7 cells in response to purified epothilone
The apoptosis of MCF-7 cells was measured by Annexin V-FITC Apoptosis Detection Kit (Cat.#K101-25), relied on the externalization of the membrane phosphatidylserine (PS) to the outer surface by the initiation of apoptosis process.The PS can be stained with the Annexin V-FITC, forming Annexin-PS conjugate that was detected by flow cytometry.The MCF-7 cells were seeded to 96-well plate (2 × 10 5 cell per well), treated with the IC 50 concentration of the purified epothilone.After incubation for 48 h at 37 °C, the cells were harvested, washed, mixed with 200 μl 1X Annexin-binding buffer, followed by Annexin V-FITC and PI, incubated in dark for 15 min at ambient temperature.Prior to the flow cytometric analysis, Annexin binding buffer was amended, and the emission of reaction was measured at λ 530 nm with excitation λ 488 nm, using FITC signal detector.

Analysis of cell cycle of MCF-7 due to the purified epothilone
The cell cycle of MCF-7 due to the putative epothilone was assessed by propidium iodide (PI) Flow Cytometry Kit assay (Cat#.ab139418).The 48-well plate seeded with the tumor cells, were incubated overnight at 37 °C, amended with purified epothilone at its IC 25 value, and re-incubated at the standard conditions.The cells were collected by centrifugation at 2000 rpm, fixed in 70% ethanol for 2 h at 4 °C, hydrated with PBS, and stained with PI for 30 min in the dark.The cellular DNA contents was checked by flow cytometry at excitation λ 493 nm and emission λ 636 nm, and the percentage of G0-G1, S and G2-M was determined by FlowJ software.

Bioprocess nutritional optimization of A. niger to maximize their epothilone productivity with response surface methodology
The physicochemical parameters including; sucrose, maltose, lactose, soytone, ammonium tartrate, yeast extract, sodium acetate, peptone, cysteine, methionine, phenylalanine, glycine, sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, calcium chloride, methyl jasmonate, in addition to fluconazole, were screened for epothilone production by A. niger with Plackett-Burman design [23,25,29].Nineteen variables were symbolized by high (+ 1) and low (− 1) levels for epothilone production by Plackett-Burman design, that follows the first ordered reaction: Y = β0 + ΣβiXi, where Y is the yield of predicted epothilone, Xi, βi and β0 are the independent variable, linear coefficient, and model of intercept, respectively.Triplicates of each run were conducted and the mean of epothilone response was considered.The significant independent variables affecting epothilone yield by A. niger from Plackett-Burman design were optimized with the face-centered composite design (FCCD) to assess their individual interactions on the epothilone yield.In FCCD, each variable was epitomized by five levels, low (− 1), medium (0), and high (+ 1), at five times center points repeats, resulting in a total 31 runs.After incubation, the epothilone yield was determined as described above.For the FCCD, a second-ordered polynomial model was used for expecting the epothilone productivity according to equation βi, βii, and βij are the regression coefficient of variables, square effects, and interactions, respectively.

Fungal deposition
The ITS sequence of A. niger EFBL-SR was deposited to the Genbank with accession # OR342867, in addition, the isolate was deposited at Assiut University Mycological Center, Egypt, with deposition # AUMC14195.

Statistical analysis
Biological triplicates of each experiment were conducted, and epothilone yield was expressed by the means ± standard deviation (SD).The F-test and statistical significance were determined by one-way ANOVA.

Isolation and screening for epothilone production by the endophytic fungi of L. loddegesii
Seven endophytic fungal isolates of L. loddegesii were isolated on PDA and morphologically examined relied on their macroscopical and microscopical characteristics, following the universal keys of identification (Fig. 1).The recovered endophytic fungi were identified as Aspergillus terreus, A. niger, A. fumigatus, Fusarium solani, Penicillium chrysogenum, P. exapnsum and Cladosporium sp.Theses isolates were grown on PDB medium at standard conditions, epothilone was extracted from the cultures, and measured by HPLC.The spots that have the same rate of mobility and color of authentic Epothilone B, as visualized at λ 254 nm, were used as the assumed epothilone spots, and measured by ImageJ software.From the TLC (Fig. 1), the highest yield of epothilone was recorded for A. niger (140.21μg/L), followed by P. chrysogenum (65.21 μg/L), while the other fungal isolates had undetectable epothilone producing potency.The target spots of silica gel were scraped off, and epothilone was extracted, and further quantified by HPLC for A. niger and P. chrysogenum as the most potent fungal isolates.From the HPLC chromatogram (Fig. 1E), a sharp peak at retention time 3.3 min was obtained from TLC purification of epothilone extracted from A. niger and P. chrysogenum, corresponding to authentic one, revealing the efficiency of the elution process of epothilone from the TLC silica gel.From the HPLC, the yield of epothilone of A. niger and P. chrysogenum were about 140.21 μg/L and 65.21 μg/L, respectively, compared to the concentration and area under the peak of the authentic one, that being identical to the results of the TLC.Thus, the yield of epothilone of A. niger and P. chrysogenum from the TLC and HPLC being consistent, confirming the efficiency of fractionation and elution processes.So, relied on the yield of epothilone, A. niger has been used for further studies.

Molecular identification, PCR mining of epothilone biosynthetic genes of the potent fungal isolate
The morphological identity of A. niger, as the potent epothilone producer from L. loddegesii, was recorded by growing on Czapek's-Dox agar and PDA media, and the macro and microscopical features were inspected daily along 10 days of incubation.The emerged fungal colonies displayed a whitish grayish color initially, which later transformed into black within a span of four days.These colonies exhibited smooth-colored conidiophores, biseriate strigma, with radial conidial heads of 3.5-5.0mm in size and vesicles ranging from 45 to 80 μm, as revealed from SEM (Fig. 2).Practically, the morphological features of this isolate, typically follows those of Aspergillus niger [62,66].The identity of A. niger was verified from their ITS sequence, the PCR amplicons of 650 bp were sequenced (Fig. 2).From the non-redundantly search BLAST, the sequence displayed 99.73% similarity and zero E-value with the ITS sequence of A. niger MW290494.1,MT609916.1,MT588789.1,MH591449.1,KP131626.1,LC634604.1,OM758326.1,KT826645.1,OQ836666.1,MN069568.1,MG590099.1,and MN944730.1.The ITS sequence of A. niger EFBL-SR was deposited to the Genbank with accession # OR342867.The phylogenetic analysis of A. niger EFBL-SR by alignment with the database deposited ITS sequences of A. niger was shown in Fig. 2.
The biosynthetic potency of A. niger epothilone was assessed determined based on the molecular expression of epoC, as an epothilone rate-limiting gene.The epoC amplicon of A. niger ~ 450 bp, was purified and sequenced (Fig. 2).The epoC sequence was translated to their corresponding protein with ExPASy tool (https:// www.ebi.ac.uk/ Tools/ st/ emboss_ sixpa ck).The phylogenetic relatedness of epoC protein was prepared by MEGA-X software, gave ~ 50% identity with uracil phosphoribosyl transferase, 3S5W ornithine hydroxylase, 6YWE Neurospora crassa, and formylase (Fig. 2).So, from the chromatographic analysis and PCR mining of the epothilone ratelimiting gene epoC, the metabolic potency of A. niger for epothilone production has been authenticated.

Chemical structure of the purified A. niger epothilone
The putative A. niger epothilone was identified by the UV-Vis analysis, FT-IR, and LC-ESI-MS/MS analyses.The UV-Vis spectra epothilone had the maximum absorption at λ 250 nm, that was identical to previously reported data of epothilone B [29,40] (Fig. 3).The FTIR absorption spectra (Fig. 3) revealed the incidence of OH groups at 3435 cm -1 and C = O groups at 1665 cm -1 .The aliphatic CH stretch was assigned to the peaks observed at 2875 cm −1 , whereas the peaks at 1641-1412 cm -1 refers to the stretch of the ester group.Additionally, an epoxy ring at 1250 cm -1 and an aromatic ring stretch at 1086 cm -1 were detected.The functional groups of the putative epothilone from A. niger were found to be consistent with previously reported data of epothilone B [29].
The molecular chemical identity of the putative epothilone of A. niger was also assessed from the LC-ESI-MS/MS analysis.From the LC-ESI-MS/MS analysis, the molecular mass of parent molecule of putative epothilone of A. niger was resolved at precursor ion [M + H] + 508.33 m/z.The molecular structure was further authenticated from the MS/MS, giving fragment ions of 313, 225 and 178 m/z (Fig. 3), that being matched with the fragmentation pattern of reported data [29].Thus, collectively from the results of UV analysis, HPLC, FTIR and LC-ESI-MS/ MS, the purified sample had identical spectral properties, molecular, and chemical structure of epothilone B [29,40,53].

Anticancer and antifungal activities of A. niger ethyl acetate extract and epothilone
The activity of A. niger crude ethyl acetate extract and epothilone towards the MCF-7, HepG-2, and HCT-116 carcinoma cell lines, compared to Vero normal cells, was determined by the MTT assay.The results of cell viability assay (Fig. 4) showed that epothilone from A. niger had a resilient activity towards MCF-7 cells (0.022 μM), HepG-2 cells (0.037 μM), and HCT-116 cells (0.12 μM) cells, compared to Vero cells (0.48 μM).These results indicated that, the extracted epothilone displayed a substantial activity against the MCF-7 (~ 22 folds) and HepG-2 cells (15 folds), compared to the normal Vero cells, that being reasonable since the rate of cellular growth of tumor cells is about 40 folds higher than the normal cells.The selectivity index of putative epothilone towards MCF-7, HepG-2 and HCT-116 was about 21.8, 12.9 and 4 folds, respectively, normalized to Vero cells (Table 1).Additionally, the efficiency of ethyl acetate extract of A. niger for the different cell lines were assessed.From the IC 50 values (Fig. 4) the crude ethyl acetate extract of A. niger exhibited a strong activity towards the MCF-7 (13.2 μg/ml), HepG-2 (19.12 μg/ ml), and HCT-116 (77.8 μg/ml), compared to Vero cells (175 μg/ml).The selectivity indices of the crude ethyl acetate extract of A. niger towards MCF-7, HepG-2 and HCT-116 cells were 13.2, 9.1 and 2.2 folds, respectively.
Practically, the selectivity index of purified epothilone towards the MCF-7, HepG-2 and HCT-116 cells was increased by about 2 folds than crude ethyl acetate extracts of A. niger.Interestingly, from the IC 50 values, the activity of epothilone was higher than that of A. niger ethyl acetate extracts by > 500 folds ensuring the efficiency of the purified epothilone.
The activity of ethyl acetate extracts of the recovered endophytic isolates of L. loddegesii were assessed against A. flavus "model human fungal pathogen".As revealed from the inhibition zones (Fig. 4C), the ethyl acetate extracts of A. niger, P. chrysogenum, P. expanum, A. fumigatus and F. solani showed the highest inhibition zones of 25.1, 22.6, 20.7, 19.2 and 18.5 mm,  respectively, compared to fluconazole as a reference antifungal compound.Furthermore, ethyl acetate extracts of each endophytic fungal isolate were implemented to assess their minimum inhibitory concentration (MIC) for A. flavus.From the results (Fig. 4), the potent -epothilone producing fungal isolate "A.niger" had the most significant activity against A. flavus, in a concentration-dependent pattern, compared to fluconazole as a positive control.Practically, the antifungal activity of the crude ethyl acetate extracts was completely matched with their constituents of putative epothilone, partially ensuring that the antifungal activity might be related to the crude epothilone polyketides.

The anti-wound healing activity of A. niger epothilone to the tumor cells
The anti-wound healing activity of the MCF-7 and HepG-2 cells due to the purified epothilone of A. niger was determined by assessing the gap closure after 72 h, compared to the negative control cells.From the data (Fig. 5), the gap closure of the HepG-2 and MCF-7 was significantly repressed with A. niger epothilone, compared to the untreated cells.The wound closure of the monolayer of HepG-2 cells was ~ 54.07 and 55.8% in response to purified epothilone of A. niger after 24 h and 72 h, respectively, compared to control.Furthermore, the gap closure of the MCF-7 tumor cells was found to be 60 to 62%, after 24-72 h, respectively, with the addition of A. niger epothilone.On the other hand, upon addition of the putative epothilone from A. niger, the wound healing of HepG-2 and MCF-7 was reduced by ~ 40-45% after 24 and 72 h, compared to the corresponding control cells, ensuring the metabolic potency of the purified epothilone for preventing the regeneration of the tested tumor cells, with subsequent ceasing to the cellular metastasis.For the higher antiproliferative Fig. 5 Wound healing assay of the HepG2 and MCF-7 cells in response to the purified epothilone of A. niger comparing to the untreated cell lines (control).After 24 h of growth of cells as a homogenous monolayer, a scratch was made, the extracted epothilone was amended to the medium at its IC50 value, the wound healing of the cell lines HepG2 (A) and MCF-7 (B) cells was measured at zero time and after 24 h and 72 h of incubation.The percentage of the wound healing as revealed from the gap area of HepG-2 (C) and MCF7 (D) cells in response to epothilone treatment was measured.The statistical analyses were conducted with one-way ANOVA, and the data were expressed by the mean ± SD efficiency of the purified epothilone of A. niger towards the MCF-7 than the HepG-2, the cell cycle and apoptosis of MCF-7 cells were further analyzed.

Cell cycle and apoptotic analysis of MCF-7 cells due to A. niger epothilone
The cell cycle of MCF-7 with the treatment of purified epothilone of A. niger was explored by the propidium iodide assay, by amendment with the epothilone at the IC 25 .The cellular DNA contents were checked by flow cytometry, and percentage of G0-G1, S and G2-M was calculated.Obviously, the maximum growth arrest of MCF-7 was measured by about 24.8% at G2/M phase, with treatment by epothilone, compared to control (Fig. 6).There is no significant difference between the G0-G1 and S phase cell cycle of the MCF-7 with the epothilone treatment and control.Obviously, the MCF-7 tumor cells were highly sensitive to the epothilone treatment at their G2/M phase, as reflected from their maximum cellular arrest.
The impact of the extracted A. niger epothilone on inducing of apoptosis of the MCF-7 cells, was evaluated by Annexin-PI assay.A significant induction to the apoptotic process of the cells of MCF-7 to early and late apoptotic stages, due to treatment by A. niger epothilone, compared to the control (Fig. 6).From the flow cytometry data, the early apoptosis, late apoptosis, and necrosis of MCF-7 cells were recorded by ~ 22.7, 7.7 and 2.6%, respectively, due to the treatment with A. niger epothilone.However, the early apoptosis, late apoptosis, and necrosis of the control cells were represented 0.57, 0.13 and 1.59%, respectively.Conclusively, the total apoptosis processes of MCF-7 cells were induced by ~ 13 folds, upon treatment with A. niger epothilone, comparing to untreated cells.Thus, from the viability assay, antifungal activity, wound healing activity, cell cycle, and apoptosis, the biological activity of the purified epothilone of A. niger has been approved, compared to the authentic one.

Bioprocessing nutritional optimization of A. niger for maximizing their epothilone yield by response surface methodology
The productivity of epothilone was enhanced by the nutritional optimization of A. niger by the response surface methodology approach.So, the chemical constituents of the medium are one of the crucial factors influencing the biosynthetic process of bioactive secondary metabolites, have been successively evaluated by the response surface methodology approaches [21,31,23,25,34].Nineteen variables involving different carbon sources, nitrogen precursors, growth elicitors were evaluated on the productivity of epothilone by A. niger.The lowest (− 1) and highest (+ 1) levels of the tested variables with the Plackett-Burman design were summarized (Table S1).The cultures of A. niger was prepared according to the designed matrix of Plackett-Burman model for 15 days at 30 °C, then the culture was filtered, epothilone was extracted and quantified.The actual and predicted response of epothilone by A. niger obtained from the Plackett-Burman design were summarized in Table 2.The statistical analysis of Placket-Burman design revealed the significance of the model as shown from the F-value (9.04), with 0.05% noise (Table S2).The model significance was provided from Prob > F" that was lower than 0.05.The "Predicted R-Squared" of 0.517 is in a rational coincidence with "Adj.R-Squared" of 0.679, the Adeq.Precision signal to noise ratio was 10.01 suggesting the adequacy of the signal (Table S2).The actual and predicted yield of epothilone by A. niger exhibited a significant variation, ranging from 22.1 to 258.1 μg/L, approving the significance of the studied variables on epothilone productivity.The Plackett-Burman design was found to be efficient, with the coefficient of determination (R 2 = 0.78), revealed the goodness of the linear regression model.The fluctuation in epothilone productivity could be due to the chemical identity of the selected independent variables, with the remaining variations accounting for only 0.01%.The results were analyzed by ANOVA, the resulting coefficients, p-value, t-Stat, and confidence levels were documented in Table S2.Pareto chart, probability, actual and predicted yield plots of the variables on epothilone productivity by A. niger were shown in Fig. S1 A-C.From the response analysis, 6 independent factors; maltose (X1), lactose (X2), methionine (X11), sodium nitrate (X13), magnesium sulfate (X15), and fluconazole (X17), have a positive influence on epothilone production.The highest epothilone productivity (258.14 μg/L) by A. niger was observed at run # 10, while the lowest yield (22.15 μg/L) was recorded at run #12.The orientation of residuals around the diagonal line indicates the independent variables distribution, ensuring the perfecting of the predicted and actual values of epothilone response (Fig. S1 D-F).ANOVA analysis reveals the significance of the constructed model, as evidenced by Fisher's F-test value 3. The maximum actual yield of epothilone by A. niger was 258.14 μg/L, with the predicted yield of 222.3 μg/L, has been reported at run #10.The highest yield of epothilone was achieved at higher concentrations of maltose (6 g/L), lactose (6 g/L), peptone (8 g/L), soytone (8 g/L), and cysteine (10 g/L) after 15 days of incubation at 30 °C.With the Plackett-Burman design bioprocessing, the epothilone response by A. niger was enhanced by ~ 1.9 folds (258.14 μg/L), compared to control culture of A. niger (140.2 μg/L).The significant parameters controlling epothilone production by A. niger were optimized by the FCCD.The most potent parameters "maltose, lactose, L-methionine, NaNO 3 and fluconazole" were tested at 5 levels, to assess their interactions on epothilone biosynthesis by A. niger.From the FCCD results (Table 3), the highest productivity of epothilone by A. niger (266.9 μg/L) was recorded at run #15, i.e. by ~ 1.9 increments folds over the control.So, the optimum medium components for maximum epothilone productivity by A. niger was maltose (1.0 g/L), lactose (2.0 g/L), L-methionine (4.0 g/L), NaNO 3 (0.1 g/L) and fluconazole (1.0 g/L).The variables of p-value < 0.1 refer to a significant, with 90% confidence, r 2 value 97.8%, ensuring the goodness of regression model.

Biosynthetic stability of epothilone by A. niger with preservation, and addition of different L. loddegesii extracts
The stability of epothilone productivity by A. niger was determined by storage at 4 °C, along 10 months, then measuring the fungal epothilone yield by the TLC and HPLC, compared to the zero culture at desired conditions.From the data (Fig. 7), a noticeable reduction to epothilone productivity has been observed with the storage, after 6 months of storage, the yield of epothilone was reduced by two folds (106 μg/L), compared to the zero culture of A. niger (258 μg/L).However, after 10 months of storage at 4 °C, the yield of epothilone by A. niger was reduced by ~ 4.8 folds (58.5 μg/L), compared to the zero culture (258 μg/L).The overall reduction of the epothilone productivity by A. niger, was similar to the previous results reporting the attenuation of productivity of the secondary metabolites "non-ribosomal peptides, polyketides, and terpenoids" with the fungal storage [29].
The impact of various organic extracts; ethanolic, dichloromethane and ethyl acetate extract of L. loddegesii leaves on restoring the productivity of epothilone by A. niger, was determined.The 4 days pre-culture of 6-months stored A. niger was amended with the leaf extracts of L. loddegesii, incubated at the desired conditions, then epothilone productivity was determined.From the results (Fig. 7), the epothilone productivity of the 6 months stored A. niger (105.1 μg/L) was partially restored by ~ 1.6 folds upon addition of L. loddegesii ethyl acetate extract (167.6 μg/L), followed by dichloromethane and ethanolic extracts.Thus, the partial restoring of epothilone productivity by A. niger with the extracts of ethyl acetate could be attributed to the chemical identity of the extracted compounds that relied to the solvent polarity.

Discussion
Epothilones have been firstly reported to be produced by S. cellulosum, with a potent anticancer activity for the multiple-drug-resistant tumors.The high water solubility of epothilones represents a pharmaceutical advantage as they don't require adjuvant for formulation and delivery [8,14].The remarkable efficacy of epothilone elaborates from its affinity to bind to β-tubulin of microtubules α, β-tubulin dimer, causing microtubule polymerization, inducing the apoptosis [54].However, the lower yield of epothilone by S. cellulosum, and the very slow growth rate are the challenges for commercial production of this compound [12,41].Several trials have been devoted to increase the yield of epothilone by S. cellulosum such as nutritional optimization [12,47], chemical modification of the media to mitigate the extracellular metabolites [64], heterologous expression of the epothilone biosynthetic genes on Streptomyces coelicolor [35,47].Recent studies have successfully enhanced epothilone production by manipulating molecular expression using TALE-TF and CRISPR/Cas9 techniques [75].Researchers are also investigating the metabolic potential of endophytic fungi and optimizing fermentation media to achieve consistent and high yields [29].These advancements offer promising prospects for industrial epothilone production.However, reduction of epothilone productivity with the fungal preservation, is the major obstacle that halts their further implementation [29].Accordingly, screening for a novel isolates inhabiting medicinal plants could be an alternative strategy to get a metabolically stable epothilone producing fungal isolate.Latania loddegesii, is one of the species of the family Arecaceae, however, the endophytic microbial flora of this plant remains ambiguous.So, the aim of this study was to isolate and identify its fungal endophytes, with a particular emphasis on their potential to produce epothilone.Among the identified endophytic fungi of L. loddegesii, A. niger OR342867.1 was the potent epothilone producer (140.2 µg/L).Moreover, the metabolic epothilone biosynthetic machinery of A. niger has been confirmed from the PCR mining of the epoC gene as rate-limiting gene of epothilone biosynthesis.PCR mining for the rate-limiting genes encoding the bioactive metabolites has been recently considered as authenticated tool to confirming the metabolic potency, by the spectroscopic and chromatographic approaches [21,31,23,26,27,29].The amplicons of epoC of A. niger exhibited 60% similarity with PKS-NRPS domains that encode the synthesis of polyketides and non-ribosomal peptides, confirming the involvement of this machinery in the synthesis of epothilones.Similarly, molecular markers of the rate limiting biosynthetic genes of epothilone [29], Taxol [49,74] and camptothecin were reported [33,60].
Aspergillus species genome mining revealed the presence of ~ 40 cryptic biosynthetic gene clusters for secondary metabolites, with 60% of these clusters being essential for the polyketide synthesis, 20% for the non-ribosomal peptide synthesis, and 40% for alkaloids compounds [10].
The chemical identity of the putative epothilone of A. niger has been confirmed from the TLC, HPLC, UV-Vis, FT-IR and LC-ESI-MS/MS analyses.The putative epothilone of A. niger exhibited the same structural identity, functional groups, molecular fragmentation pattern (508.33 m/z) coincident with the reported data of epothilone B [29,40,53].
The antiproliferative activities of the extracted epothilone and crude ethyl acetate extract of A. niger towards the MCF-7, HepG-2, HCT-116 cells, compared to Vero normal cells, were assessed.The antiproliferative activity of the putative epothilone towards the MCF-7 and HepG-2 cells, compared to the normal Vero cells was increased by ~ 22 and 15 folds.These findings were reasonable since the rate of cellular proliferation of tumor cells is about 40 folds higher than normal cells.The selectivity indices of putative epothilone to MCF-7, HepG-2 and HCT-116 were ~ 21.8, 12.9 and 4 folds, respectively, compared to the Vero cells.The antiproliferative activity of the purified epothilone of A. niger was relatively higher than that of A. fumigatus [29].The superior antiproliferative activity of the purified epothilone compared to Taxol on various cell lines can be attributed to its high water solubility and its ability to target Taxol-resistant tumor cells at a molecular level [8,42].The ethyl acetate extract of A. niger had a strong activity towards the MCF-7, HepG-2 and HCT-116, compared to the Vero cells, with selectivity indices 13.2, 9.1 and 2.2 folds, respectively.The Fig. 7 Biosynthetic stability of epothilone productivity of A. niger in response to storage and addition of plant extracts.A The epothilone productivity by A. niger with the fungal preservation as slope culture (The upper panel is the TLC, the lower panel is the yield epothilone).B The yield of epothilone by A. niger in response to amendment with different extracts of Latania loddegesii.The statistical analyses were conducted with one-way ANOVA, and the data were expressed by the mean ± SD antiproliferative activity of the purified epothilone of A.
was dramatically increased by ~ 500 folds compared to their ethyl acetate extracts, ensuring the specificity of the purified epothilone for inhibiting the tubulin polymerization [42].The anticancer activity of the purified epothilone of A. niger closely matched that reported for epothilone from A. fumigatus, toward the different cell lines [29], ensuring the similar structural activity relationship of epothilone from the different fungi.The highly epothilone producing fungal isolate "A.niger" had the most significant activity against A. flavus, in a concentration-dependent manner, compared to fluconazole, with IC 50 values 17.7 μg/ml.The crude ethyl acetate extracts of the recovered fungal isolates exhibited a full alignment of antifungal activity with their putative epothilone constituents, indicating that their effectiveness may be attributed to the presence of crude epothilone.The potential of a drug to exhibit both anticancer and antifungal activities is highly promising, especially considering the susceptibility of immune-compromised patients to microbial infections.Therefore, the development of a drug that possesses dual properties of combating cancer and microbial infections holds great potential and affordability [37].Invasive Aspergillosis (IA) with A. flavus and A. fimgatus exhibits a higher incidence compared to invasive candidiasis (IC) [4,58].Aspergillus flavus is the main cause of invasive fungal rhinosinusitis, otitis, keratitis and lung infections in cancer patient with immunodeficiency [45], with a noticeable resistance to the common antifungal drugs.In addition, A. flavus has been frequently associated with various clinical syndromes, notably chronic granulomatous invasive fungal sinusitis, cutaneous aspergillosis, wound infections, keratitis and osteomyelitis resulting from trauma and inoculation [4,58].Similarly, the ethylacetate extract of A. tubingensis, an endophyte of Malus domestica, had an obvious antifungal activity towards Fusarium solani and A. niger [55]. A. niger, an endophyte of Nigella sativa seeds, had a high amount of phenolic compounds and flavonoids with a noticeable antioxidant activities [61,2].Consistently, the ethylacetate extracts of Penicilluim commune, A. flavipes and Fusarium chlamydosporum had a remarkable antioxidant activity, by scavenger the different free radicals [61,2].
With the purified epothilone, the wound healing of HepG-2 and MCF-7 cells was reduced by ~ 40-45% after 24 h, compared to the control cells, confirming the suppression of the cellular regeneration, matrix formation of the tumor cells, with an actual subsequent ceasing to the cellular metastasis [78].The cellular growth arrest of MCF-7 cells was maximally observed by 24.8% at the G2/M phase with A. niger epothilone treatment, compared to control cells.The efficiency of epothilone to inhibit the cellular growth at the G2/M phase has been frequently reported [38,65], ensuring the proximity of biological identity of the purified epothilone with the reported one.Additionally, a significant induction to the apoptotic process of the MCF-7 cells, in response to treatment with A. niger epothilone, has been detected, by ~ 13 folds, compared to control cells.Conclusively, from the chemical analysis and biological activities the purified putative compound of A. niger has been confirmed as epothilone B.
The epothilone productivity by A. niger was maximized by the nutritional optimization bioprocessing with the Plackett-Burman Design, since the medium components and their interactions had a significant effect on the biosynthesis of the fungal bioactive secondary metabolites [31,[23][24][25][26]29].The yield of epothilone by A. niger was increased by about 1.9 folds, compared to control culture (140.2 μg/L).Similar optimization pattern using the surface response methodology for maximizing the yield of epothilone, Camptothecin and Taxol production by fungi has been reported [31, 23-26, 29, 34].Interestingly, the yield of epothilone by the current isolate A. niger (258.1 μg/L) was quite higher than that of A. fumigatus (60 μg/g biomass) [29], and Burkholderia DSM7029 (8.1 μg/l) [51].Nevertheless, the yield of Epothilone B by the nutritionally optimized Sorangium cellulosum was about 39.7 mg/L [52,57], that being relatively higher than the current isolate of A. niger.However, for the feasibility of mass production under submerged conditions and rapid growth rate, A. niger being more affordable approach than S. cellulosum for production of Epothilone B. Since, S. cellulosum is usually of biological slow growth, dependence on growth density, easily to contaminate, instability, and sensitivity to the environment conditions, that subsequently causing its extraordinarily high price [44,57].In addition, S. cellulosum usually form a biological clump that hinders their nutrient uptake with a subsequent suppression of secondary metabolites productivity, under submerged conditions, so, these bacteria usually grow on solid medium in presence of resin to scavenger the released metabolites [40].Biologically, these bacteria have a complex living pattern by feeding in groups, moving in swarms, and developing multi-cellular fruiting bodies [67], their metabolites is strongly affected by the coexisting other microorganisms [51].
A noticeable reduction to the epothilone productivity has been observed with the storage of A. niger, the yield of epothilone was reduced by about 2.4 folds compared to the zero culture of A. niger, after 6 months.Consequently, this reduction of epothilone productivity by A. niger, was the challenge that halts its further exploitation for industrial applications, as consistent with various secondary metabolites "non-ribosomal peptides, polyketides, and terpenoids" with the fungal [49].Consistently, reprograming the cellular biosynthetic machinery of secondary metabolites by the endophytic fungi to suppress their overall productivity is the common metabolic traits, with the storage and subculturing of the authentic fungi [31,23,29].The loss of the biosynthetic potency of the secondary metabolites by fungi could be related to the derived signals from the host plant, and/or microbialmicrobial interaction, cross-talk of the different microorganisms [26,29].Thus, impact of various organic extracts namely ethanolic, dichloromethane and ethyl acetate extract of L. loddegesii leaves on restoring the epothilone productivity by A. niger has been evaluated.Remarkably, the epothilone productivity of 6 months stored A. niger was partially restored by ~ 1.6 folds with L. loddegesii ethyl acetate extract.This partial restoring upon using of plant ethyl acetate extracts, relatively negates the association of plant-derived signals, referring to the hypothesis of fungal-microbiome interactions that might has the direct effect on induction of the cryptic genes of epothilone biosynthesis [23,34].
In conclusion, the isolate A. niger "an endophyte of L. loddegesii" had the highest potency for epothilone production.The chemical structure of the purified A. niger epothilone was resolved by LC-ESI-MS/MS, compared to the reported data.The purified epothilone from A. niger had a strong activity against HepG-2, and MCF-7 cell lines, with high selectivity index compared to normal Vero cells, and highest activity as anti-wound healing of the MCF-7 cells.The epothilone had a strong activity in arresting the growth of MCF-7 cells at G2/M phase.The productivity of epothilone by A. niger was increased by ~ 1.8 folds, with the response surface methodology.However, the yield of epothilone by A. niger was reduced sequentially by ~ 2.4 folds with the fungal storage for 6 months, with a relative restoring to the biosynthetic machinery with addition of ethyl acetate extract of L. loddegesii.So, with further proteomics and transcriptomics analysis to explore the metabolic machinery of epothilone biosynthetic regulation, A. niger could be a novel platform for industrial production of epothilone B.

Fig. 1
Fig. 1 Isolation and screening for epothilone production from the endophytic fungi of Latania loddegesii leaves.A Morphological view of Latania loddegesii.B Plate cultures of the recovered fungal endophytes.C TLC of the extracted epothilone from the fungal isolates normalizing to authentic epothilone.D Epothilone yield as quantified from the TLC chromatograms.E HPLC chromatogram of the purified epothilone from the most potent isolates, normalized to the authentic one, with retention time 3.3 min.The statistical analyses were conducted with one-way ANOVA, and the data were expressed by the mean ± SD

Fig. 2
Fig. 2 Identification of the most potent epothilone producing isolates A. niger from L. loddegesii.The plate culture and conidial heads of A. niger (A, B).C Gel of gDNA and PCR amplicon of the ITS region.D The molecular phylogenetic relatedness of A. niger based on the sequence of its ITS region by the Maximum Likelihood method.E PCR amplicons of A. niger epoC, the amplicons were purified, sequenced, and in silico translated to their corresponding proteins by ExPasy portal.F The phylogenetic analysis of deduced amino acid sequences of epoC of A. niger by Mega 7.0 Software

Fig. 3
Fig. 3 Chemical identity of the putative epothilone from A. niger.The putative epothilone spots were scraped off from the TLC plats based on their color and mobility, normalized to the authentic one.The UV-spectra (A) and FT-IR chromatogram (B) of the putative epothilone.C UPLC-ESI-MS/MS analyses of the purified Epothilone with the onset chemical structure of Epothilone B

Fig. 4
Fig.4 The antiproliferative and antifungal activities of the extracted epothilone and crude ethyl acetate extracts of A. niger.The antiproliferative activity of the putative epothilone (A), and crude ethyl acetate extracts (B) towards the tumor cell lines HepG-2, MCF-7, and HCT-116, compared to the normal Vero cells, a revealed from the IC 50 values.C The diameter of the inhibition zones of the ethyl acetate extracts of the tested fungal extracts against A. flavus.D The antifungal activity of the extract Epothilone and crude ethyl acetate extracts of A. niger against A. flavus at concentrations 0.1, 0.5, 2.5, 5 and 10 μg/ml.The statistical analyses were conducted with one-way ANOVA, and the data were expressed by the mean ± SD

Fig. 6
Fig.6 Flow cytometric apoptotic analysis of the MCF-7 cells by Annexin V-FITC.The cells were treated with the extracted epothilone at their IC 25 , the apoptosis was measured after 48 h of incubation.The cell cycle of the control MCF-7 cells (A), treated with epothilone (B), and overall cell cycle growth arrest analysis (C).The apoptotic analysis of the control MCF-7 cells (D), treated with epothilone (E), and the overall apoptosis percentage (F).The statistical analyses were conducted with one-way ANOVA, and the data were expressed by the mean ± SD

Table 1
The antiproliferative activity of the crude ethyl acetate extract and the putative epothilone from A. niger against MCF-7 and HepG-2, HCT-116 carcinoma cell lines and VERO normal cell line CC 50 (50% cytotoxic concentration) and IC 50 (50% inhibitory concentration) values are expressed; SI (selectivity index) = CC 50 value of normal cell/IC 50 value of cancer cell As revealed from the IC 50 value, ethyl acetate extracts of A. niger and P. chrysogenum had the highest potency against A. flavus with IC 50 values 17.7 and 19.2 μg/ml, respectively.While the IC 50 values of ethyl acetate extracts of P. expansum and A. fumigatus was approximately 21.5-23.4μg/ml.

Table 2
Matrix of Plackett-Burman experimental design for epothilone B production by A. niger

Table 3
Matrix and responses of the CCD for the significant factors affecting Epothilone B production by A. niger